Endocytosis of opioid receptors modulates signal transduction and is thought to be involved in opiate drug action and addiction. This process is highly regulated by ligands, both in cultured cells and native tissues. A phosphorylation-dependent mechanism that promotes receptor endocytosis has been described previously. Preliminary studies suggest the hypothesis that additional mechanisms contribute to the physiological regulation of opioid receptor endocytosis observed in intact cells. Furthermore, the preliminary data suggest that these mechanisms are relevant to opiod receptor regulation in neurons. The proposed studies test this hypothesis by focusing on two distinct mechanisms, which have opposite effects on receptor endocytosis and differ in their dependence on receptor phosphorylation. The proposed experiments seek to elucidate these mechanisms in biochemical detail, identify cellular proteins that mediate them, and examine their functional consequences in neurons. The Specific Aims are to (1) characterize a mechanism that promotes rapid endocytosis of a truncated, phosphorylation-deficient mutant opioid receptor; (2) characterize a phosphorylation-regulated mechanism that inhibits endocytosis of full-length opioid receptors; and (3) identify cellular proteins that interact specifically with defined receptor domains and elucidate their role in regulating endocytosis of opioid receptors in several cell types. These studies have direct relevance to mechanisms underlying opiate drug action and addiction. In addition, they may be relevant to understanding alternate mechanisms regulating endocytosis of other G protein-coupled receptors.